KR20140000107A - Novel bacteriophage and its use for preventing proliferation of aeromonas hydrophila - Google Patents

Abstract

The present invention relates to a novel bacteriophage which is newly isolated and identified and to a composition for suppressing proliferation or killing Aeromonas hydrophila which includes the novel bacteriophage as an active ingredient. The present invention can be variously utilized in a composition for preventing or treating Aeromonas hydrophila infectious diseases, an antibiotic, a disinfectant, feed additives for fishes, and the likes.

Description

[0001] The present invention relates to novel bacteriophages and their use for the inhibition of aeromonas hydropila proliferation,

The present invention relates to a novel phage which has been newly isolated and identified, Aeromonas hypha pylas containing it as an active ingredient hydrophila ) proliferation inhibiting or killing composition, a composition for preventing or treating an aeromonas hydropila infectious disease, an antibiotic, a disinfectant, and a feed additive for fish.

Aeromexico on to, the movement ah belonging to monada seae (Aeromonadaceae) as a person, ah Monad (motile aeromonas) Monastir Heathrow Pilar (Aeromonas hydrophila) is susceptible to a wide variety of fish, causing sepsis, bleeding, ulcers, etc., and this Causing serious economic damage. Especially, in the case of domestic farms, the density of breeding is high due to limited space, so the occurrence of Aeromonas hydropila is highly contagious. For example, Nishikigoi and goldfish at the fish market in Jincheon, Chungcheongbuk-do are representative ornamental fish that are severely damaged by aeromonas hydropilas.

Because Aeromonas hydropila is an aquatic organism, it is often treated with antibiotics for the purpose of controlling it. As a result, most aeromonas hydropilas are resistant to antibiotics, It is not only a threat to health but also the damage caused by the emergence of resistant bacteria.

On the other hand, bacteriophage is a virus that is specifically infected with bacteria. In 1915, British bacteriologist Twort discovered in a study of the phenomenon of transparent melting of the micrococcus colonies by something. In 1917, a French bacteriologist d'Herelle discovered that there was an effect of dissolving Shigella disentriae in the filtrate of dysentery patients. Through the research, they discovered bacteriophages independently and eaten bacteria Called bacteriophage in the sense of. However, the use of antibiotics has led to the emergence of multidrug-resistant pathogens and superbacteria, and many problems with conventional antibiotics have emerged. , And has been attracting attention as an alternative to antibacterial agents. Moreover, bacteriophage has a short development time, low development cost, and superiority in terms of bacterial selectivity, persistence and side effects compared to antimicrobial agents.

Accordingly, a problem to be solved by the present invention is to separate a new bacteriophage having a lytic activity in Aeromonas hydropila, to provide a composition for inhibiting or proliferating Aeromonas hydropila, a composition for preventing or treating aeromonas hydropila infection A pharmaceutical composition for preventing or treating disease, an antibiotic, a disinfectant, and a feed additive for fish.

In one aspect, the invention relates to bacteriophage having specific killing activity against Aeromonas hydrophila .

In the morphological Mai ohbiri to (Myoviridae) and (family) kawoodo Bilal less (Caudovirales) neck (order), and

Bacteriophage of accession number KCTC 12128BP or bacteriophage of accession number KCTC 12129BP.

The present inventors have isolated and identified phage having the ability to kill Aeromonas hydrophila , named PAH1-C and PAH6-C, and on Feb. 3, 2012, No. KCTC 12128BP and Accession No. KCTC 12129BP.

The present inventors collected samples from Haesung Seocheon and analyzed the aeromonas hydropilas ( Aeromonas hydrophila) La phage lysis them the JUNAH as a host (hereinafter, PAH1-C hereinafter) the separation was phage lysis of Trichomonas dihydro pillar JUNAH to remain on from a sample collected from Nanji water reclamation center (hereinafter, PAH6-C hereinafter ). These PAH1-C and PAH6-C were found to form plaques on Aeromonas hydrophila to have bacteriolytic activity.

Further, it was found to belong to these PAH1-C and C-PAH6 a result of observation of the morphology by electron microscopy, to my ohbiri (Myoviridae) and (family) kawoodo Bilal less (Caudovirales) neck (order).

In addition, the genomes of PAH1-C and PAH6-C were analyzed and found to be composed of double-stranded DNA (dsDNA) with a genome size of about 50 kb. The entire genome of PAH1-C and PAH6- , The genomic size of PAH1-C and PAH6-C was found to be a novel bacteriophage showing a great difference from the bacteriophage known to have the ability to infect a previously reported aeromonas hydropila.

In the specification of the present invention, 'PAH1-C or PAH6-C' includes phage cultures as well as phage itself.

The phage PAH1-C or PAH6-C phage according to the present invention can be cultured in a large amount by a conventional phage culturing method. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. For example, NB (nutrient broth), TSB (tryptic soy broth) liquid medium, or TSA (tryptic soy agar) solid medium can be used. The culture can be carried out under ordinary culture conditions. The culture medium in the culture medium may be removed and centrifuged or filtered to collect only the concentrated cells, which step may be carried out according to the needs of those skilled in the art. The concentrated microbial cells can be preserved by freezing or freeze-drying according to a conventional method so as not to lose their activity.

PAH1-C and PAH6-C show susceptibility to Aeromonas hydrophila .

More specifically, PAH1-C shows susceptibility to Aeromonas hydropilas AH1, AH6, AH9, AH16, AK1 and AK9, and PAH6-C is sensitive to Aeromonas hydropilas AH7, AH16, AK1 and AK9 It shows sensitivity.

Further, preferably, the Aeromonas hydrophila is an antibiotic-resistant strain or a multidrug-resistant strain.

Accordingly, in another aspect, the present invention as the O-containing PAH1 PAH6 C or C-pillar, as an active ingredient, Pseudomonas hydrochloride (Aeromonas hydrophila ) proliferation inhibiting or killing composition or a method for inhibiting or killing the growth of aeromonas hydropilla using the same.

In another aspect, the present invention relates to PAH1-C or PAH6-C, or antibiotics, disinfectants and feed additives for fishes comprising the same as an active ingredient. Preferably, phage PAH1-C or PAH6-C, or Aeromonas hydropilas containing it as an active ingredient hydrophila ) proliferation inhibiting or killing antibiotics, disinfectants and feed additives for fish.

In the specification of the present invention, the term 'antibiotic' means a preservative, a bactericide, and an antibacterial agent.

The feed additive may be added to feed for fish.

Alternatively, PAH1-C or PAH6-C may be added directly to the feed without preparing it as a feed additive and mixing with the feed.

The fish include carp, cichlid, perch, cod, crucian carp, salmon, beetle, loach. But are not limited to, catfish or rodents.

Aeromonas hydropilas according to the present invention hydrophilia ) Antimicrobial agents, antibiotics, disinfectants, and feed additives for proliferation inhibition or killing are highly specific to Aeromonas hydrophila as compared with conventional ones, and thus it is possible to kill only specific pathogens without killing them It is possible to kill multidrug-resistant pathogens, which not only exerts excellent effects but also does not induce tolerance or resistance of pathogens, and thus has a longer product life than conventional ones.

The compositions, antibiotics, disinfectants and feed additives for inhibiting or killing aeromonas hy- drophila may be prepared in liquid form such as solutions, suspensions and emulsions, but may be formulated as powders, granules, tablets or capsules It can be produced in the same solid phase.

The compositions, antibiotics, disinfectants and feed additives for inhibiting or killing the aerosol hydropilase may further comprise an acceptable carrier and may be formulated together with the carrier. The acceptable carrier refers to a carrier that does not irritate the organism and does not interfere with its biological activity and properties. For the purpose of stable formulation suitable for actual packaging, it may be prepared in the form of a wetting agent, a granular wetting agent, a liquid wettable powder, a liquid agent, a water-soluble agent, a water-soluble granule or an encapsulating agent using a surfactant or an extender. Examples of the surfactant include alkyl (C ₈ -C ₁₂ ) aryl sulfonate, dialkyl (C ₃ -C ₆ ) aryl sulfonate, dialkyl (C ₈ -C ₁₂ ) sulfosuccinate, lignin sulfonate, naphthalene Sulfonate condensates, naphthalene sulfonate formalin condensates, alkyl (C ₈ -C ₁₂ ) naphthalene sulfonate formalin condensates, sodium salts of sulfonate compounds such as polyoxyethylene alkyl (C ₈ -C ₁₂ ) phenylsulfonate, or calcium salts, alkyl (C ₈ ~ C ₁₂₎ sulfates, alkyl (C ₈ ~ C ₁₂₎ aryl sulfates, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ sulfates, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ phenyl sulphate , Sodium salts or calcium salts of succinate compounds such as polyoxyalkylene succinates, sodium benzoates, anionic surfactants such as alkylcarboxylates, polyoxyethylene alkyl (C ₁₂ ~ C ₁₈₎ ethers, polyoxyethylene al (C ₈ ~ C ₁₂₎ phenyl ether, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ phenyl polymer and ethylene oxide propylene oxide from the non-ionic surfactants, polycarboxylate, a group consisting of Triton 100 and Tween 80, such as a copolymer It will be readily understood by those skilled in the art that one or more selected surfactants may be used in combination, and surfactants other than these surfactants may also be used. Examples of the extender include bentonite, talc, clay, kaolin, calcium carbonate, silica, pumice, diatomaceous earth, acidic clay, zeolite, pearlite, white carbon, ammonium sulfate, urea, glucose, dextrin, , Yellow loess, glucose and starch, water, etc. may be used singly or in combination of two or more, and other extender may be used, as will be readily understood by those skilled in the art will be.

It will be apparent to those skilled in the art that the composition of the composition for inhibiting or killing aeromonas hy- drophila, the antibiotic, the disinfectant, and the feed additive can be arbitrarily adjusted to suit the purpose of use, It will be easily understood.

The compositions, antibiotics, disinfectants and feed additives for inhibiting or killing aeromonas hy- drophilas may include PAH1-C or PAH6-C, i.e., only one phage, and both of the two phages may be included. In addition, the effective bacteriophage may further include an effective bacteriophage, which preferably has a lytic activity against Aeromonas hydropilas, but also includes a phage having a lytic activity against other bacteria .

In addition, when the composition for inhibiting or proliferating aeromonas hydropilas is treated, an antibiotic, a disinfectant, and a feed additive can be used in combination with a conventional method for inhibiting or killing bacterial growth including aeromonas hydropilas And preferably used in combination with a method conventionally used for inhibiting or killing aeromonas hydropilas.

In another aspect, the present invention provides a pharmaceutical composition comprising Aeromonas hypha pylas containing phage or a culture thereof as an active ingredient hydrophila ) pharmaceutical composition for preventing or treating an infectious disease, or a pharmaceutical composition for preventing or treating an infectious disease by using Aeromonas hydropilla hydrophila ) infectious diseases.

As it described above, the grip according to the present invention, so as to have the O-specific killing capability for Pseudomonas dihydro pillar (Aeromonas hydrophila), O to Pseudomonas dihydro pillar (the Aeromonas hydrophila ) infections. < / RTI >

Preferably, the aeromonas hydropilas ( Aeromonas hydrophila) infectious diseases, sepsis, but are not limited to Monastir disease or ulcers, or in Arezzo, this. In addition, the Vibrio parahaemolyticus infectious diseases also include symptoms expressed from such diseases, including, but not limited to, diarrhea or hemorrhage.

In the specification of the present invention, the term 'prevention or treatment' includes Aeromonas hydropilas hydrophila) includes the prevention, inhibition, and reduction of the infectious disease.

The composition of the present invention may be administered to an animal in the form of a pharmaceutical preparation, or mixed with feed or water and fed to the animal.

The administration route of the composition of the present invention may be administered through various routes of oral or parenteral administration as long as it can reach the target tissues. Specifically, oral administration, rectal administration, topical administration, intravenous injection, intraperitoneal injection, intramuscular injection, Transdermal, non-intranasal, inhalation, and the like.

A prophylactic or therapeutic method of the present invention includes administering a composition of the present invention in a pharmaceutically effective amount. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. The specific therapeutically effective amount for a particular subject will depend upon the type and extent of response to be achieved, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the fraction of the route of administration, It is preferable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.

The newly separated phage according to the present invention has Aeromonas hydrophila proliferation inhibition or killing effect, and particularly has a proliferation inhibiting or killing activity against antibiotic resistant Aeromonas hydropilas, Aeromonas disease or ulcers such as Aeromonas It is effective for the prevention or treatment of infectious diseases of hydropila, and it can be used as an antibiotic or disinfectant as well as being utilized as feed. Therefore, it is expected to contribute to the competitiveness of the aquaculture industry and the improvement of public health.

Figure 1 shows the results of pulsed-field gel electrophoretograms of the degradation products by PAH1-C gDNA (left) and PAH6-C gDNA (right) and 12 restriction endonucleases . The M line represents the low range PFG marker (New England BioLab). Line 1 shows the phage gDNA that was not degraded. Line 2-13 shows the phage PAH1-C and PAH6-C degradation patterns produced by SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI BioLab). The genome size of the two phage gDNAs was estimated to be about 50 kb.
FIG. 2 is an electron microscopic observation of negative stained PAH1-C (left) or PAH6-C (right).
FIG. 3 is a graph And PAH1-C and PAH6-C against AH16. Co-cultured with PAH1-C or PAH6-C with MOI (muliplicity of infection) 0.01, 1, and 100. The results are expressed as mean + standard deviation values through three tests.
Figure 4 illustrates the therapeutic effect of the in the loach Oh Pseudomonas dihydro pillar (A. hydrophila) PAH1-C and C-PAH6 gripping of the infection.

≪ Materials and methods >

Sampling of target bacteria and phage

Samples were taken from sewage treatment plants, rivers, and rivers around fish farms. After sampling the place and date, more than 500 ml was sampled and 100 ml was used for bacteriophage separation. Specifically, the bacteriophage was isolated from the Seosam Stone and the Nanji Water Reclamation Center. At the same time, bacteria were isolated from farms or rivers. Isolated bacteria were identified using PCR and Vitek II.

Separation of phage and host station investigation

(Adams M (1959) Bacteriophages. Interscience Publishers, New York), which is commonly used for phage separation and the numbering of plaque-forming units (PFUs) thereof. Respectively. Aeromonas hydrophila hydrophila JUNAH was used as a host bacterium for phage separation. The plaque morphology was observed after 18-24 hours of incubation. The separating material is incubated at 20 DEG C for 36 hours, centrifuged at 10,000 x g for 20 minutes, filtered through a 0.45 mu m filter to add 10 mL of 10 x TSB to 90 mL and incubated with host bacteria 10 ⁶ to 10 ⁷ CFU And cultured for more than 12 hours. After centrifugation at 5,000 rpm for 20 minutes, the supernatant was collected, and 10 ⁸ CFU of host bacteria was applied to the surface of the TSA agar medium. 5 ~ 10ul of the supernatant was dropped and cultured at 25 ° C for 18 hours. When lysis was observed, the plaques were observed by determining the titers by ten-fold dilution. Pure phage strains were obtained through a series of single-plaque isolations and named PAH1-C and PAH6-C. The filtered phage lysate was precipitated with 10% (wt / vol) polyethylene glycol 8000 in 1M NaCl at 4 ° C for 12 hours and recovered by centrifugation at 10,000 x g for 10 minutes at 4 ° C. The purified phages were washed with SM buffer (10-mM NaCl, 50-mM Tris [pH 7.7; 5] and 10-mM MgSO ₄ ) and stored at 4 ° C until used.

The host strains of PAH1-C and PAH6-C were prepared by adding 10 [mu] l of diluted phage suspension (3.3 [mu] l) to a double-layer agar plate inoculated with Aeromonas hydrophila and Aeromonas sorbia 10 < ⁷ > PFU / ml). The bacteria were then incubated at 37 [deg.] C for 20 hours and checked for plaque formation.

Discrete phage Genetic  analysis

Purified phage genomic DNA (gDNA) was obtained from Son JS et al. (2010) Complete genome sequence of a newly isolated lytic bacteriophage, EFAP-1 of Enterococcus faecalis, and antibacterial activity of its endoly sin EFAL-1.J Appl Microbiol 108: 1769-1779, For analysis, the phage genome response to DNase I (20 U / μl) (Takara), RNase A (5 U / μl) (intron) and Mung bean nuclease (20 U / μl) (Takara) . First, 10 ⁹ PFU / ml or more of phage stock was removed with Proteinase K, and the supernatant treated with PCI (phenol-chloform-isoamyl alcohol) was treated with isopropanol and washed with 70% ethanol And the genome was finally concentrated in a TE buffer. The enriched phage genome was electrophoresed in 0.7% agarose gel (1X TAE buffer) and used as a control. Each phage group was treated with DNase I, RNase A, and Mung bean nuclease, The results were determined by the presence or absence of.

In addition, Uchiyama J et al., (2008) Iso-lation and characterization of a novel Enterococcus faecalis bacteriophage EF24C as a therapeutic candidate for precisely observing the genome size and restriction enzyme cleavage pattern of phage gDNA. Pulsed-field gel electrophoresis (PFGE) was performed according to a slightly modified method as described in FEMS Microbiol Lett 278: 200-206. Briefly, 500 μl of phage suspension was mixed with 2% (wt / vol) NuSieve GTG agarose (FMC BioProducts), poured into a plug mold and solidified. The flock was punched through a mold into a small volume of digestion buffer (500 mM EDTA, 10 mM Tris [pH 8.0], 1% SDS [wt / vol] and 1 mg / ml proteinase K) and incubated overnight at 50 ° C . The digestion buffer was removed and the sample was washed 3 times with TE buffer and then 10 U of SacII, Sau3AI, MspI, XbaI, NotI, HindIII, SmaI, SphI, NcoI, HpaII, SpeI and EcoRI (New England Biolabs ) At 37 ° C for 1 hour, respectively. The flasks were washed three times with TE buffer and placed in wells of 1.2% Pulsed Field Certified agarose (Bio-Rad) in 0.5X TBE and covered with molten 0.5% NuSieve GTG agarose. The samples were electrophoresed with CHEF-DR III System (Bio-Rad) at 6 V / cm with a pulse lamp for 15-15 seconds at 14 ° C in 0.5X TBE buffer.

Electron microscopic analysis of isolated phage

Transmission electron microscopy (TEM) was performed for morphological classification of isolated phage. The phage stock was loaded onto a copper grid, followed by negative staining with 1% uranyl acetate and drying. Electron microscopy took place at NICEM, the College of Agriculture and Life Sciences, Gwanak Campus, Seoul National University. The morphology of the phage was observed using a Zeiss TEM EM902 (Zeiss) at a voltage of 80 kV. The phage size was calculated by measuring over 10 times.

Broth - culture  Detection of phage susceptibility to host bacteria

The lytic activity of PAH1-C and PAH6-C against Aeromonas hydrophila was evaluated. The cells were treated with PAH1-C (1.1x10 ¹⁰ PFU / ml) and PAH6-C (1.1x10 ¹⁰ PFU / ml) and Aeromonas hydropila AH16 (8.8x10 ⁶ CFU / ml) Aeromonas hydropilas ( Aeromonas hydrophila AH16 was divided into three groups: MOI (multiplicity of infection, quantitative ratio of bacteria and bacteriophage) 0.01, 1, 100 in 10 ml of clean TSB. The product was co-cultured at 20 ° C with shaking at 250 rpm. Bacteria inoculated into TSB without phage were used as controls. Absorbance (OD ₆₀₀ ) was measured until 24 h after inoculation.

Experimental Aeromonas Hydropila  Effectiveness of phage treatment by artificial infection

Loach (Cobitis biwae, average length: 14.56 cm, average weight: 14.98 g), which is known to have the highest sensitivity to Aeromonas hydropila, was used. AEROMONAS HYDROPILLA used for artificial infection was AEROMONAS HYDROPHILA strain AH16 which caused mass mortality in loach, PAH1-C and PAH6-C were used for phage. Intramuscular injection (IM, intra-muscular injection) was used and the therapeutic phage were administered in the same way. The experimental groups were divided into four groups: those not treated with phage after bacterial infection (20 percent), those treated with PAH1-C phage (20 percent) after bacterial infection, those treated with PAH6-C phage (20 percent) And PAH1-C and PAH6-C phages (20 controls). Each experiment was repeated three times to obtain more accurate results. In the case of the AH16 strain used for bacterial infection, it was diluted with PBS to a concentration of 3.3 × 10 ⁷ CFU / fish and artificially infected. The PAH1-C used in the phage treatment was 1.1 × ^{10 9} PFU / fish and the PAH ⁶ -C was 1.0 × ^{10 9} PFU / fish. Each experimental group had prognosis for 7 days at 25 ℃ water temperature.

<Result>

Separate phage and Host station

A total of 11 isolations were used to isolate the two phages. The phage were separated pure through three cloning steps. The phage named PAH1-C was isolated from Haesung Seocheon, and Aeromonas hydropilas hydrophila) to form a plaque in JUNAH. In addition, the phage named PAH6-C was isolated from the Nanji Water Recovery Center and formed plaques in Aeromonas hydrophila JUNAH .

A total of 8 separate aeromonas hydropilas ( Aeromonas hydrophila , and aeromonas sorbia ( 10 ) were investigated for susceptibility and host status of each of two kinds of phage. PAH1-C formed plaques in six of the seven Aeromonas hydropilas of Aeromonas hydropila and one of ten Aeromonas sorbia of Aeromonas sorbia. In addition, PAH6-C formed plaques in four weeks of Aeromonas hydropila in seven weeks of Aeromonas hydropila and one in Aeromonas sorbia of ten weeks of Aeromonas sorbia.

Therefore, it was judged that both PAH1-C and PAH6-C showed susceptibility to Aeromonas hydropila (Table 1). To a Table 1 corresponding to all of the Pseudomonas dihydro pillar is an antibiotic-resistant strain, especially of the O to Pseudomonas dihydro pillars for use as a host bacterium (Aeromonas hydrophila ) Aeromonas hydropila JUNAH is a multidrug-resistant strain.

Strain name
Serial Number
Separation place
sensibility
PAH1-C PAH6-C A. hydrophila AH1 Koi ( Cyprinus carpio carpio ) + - A. hydrophila AH6 Congo tetra ( Phenacogrammus interruptus ) + - A. hydrophila AH7 Sailifin molly ( Poecilia latipinna ) - + A. hydrophila AH9 Cherry salmon ( Oncorhynchus masou masou ) + - A. hydrophila AH16 Loach ( Cobitis biwae ) ++ + A. hydrophila AK1 Han Liver + ++ A. hydrophila AK9 Sewage in Gyeonggi province ++ ++ A. Sorbia AS1 Goldfish ( Carassius auratus auratus ) + + A. Sorbia AS2 Neon tetra ( Paracheirodon innesi ) - - A. Sorbia AS3 Koi ( Cyprinus carpio carpio ) - - A. Sorbia AK2 Lake - - A. Sorbia AK3 Lake - - A. Sorbia AK4 Lake - - A. Sorbia AK5 Lake - - A. Sorbia AK6 Lake - - A. Sorbia AK7 Lake - - A. Sorbia AK8 Lake - -

The separated phages were stored at 4 ° C in a dark place.

PAH1 -C PAH6 Classification and Characteristics of -C

1) Classification of phage according to dielectric properties

In general, mio viride de phage is known to have a double helix (ds) DNA genome. Similarly, the gDNAs of PAH1-C and PAH6-C are completely degraded by DNase I, but not completely degraded by RNase A or Mung bean nuclease. Therefore, it was estimated to be ds (double strand) DNA. In addition, the gDNA SacII, Sau3AI, MspI, SmaI, NcoI, was truncated by the HpaII and EcoRI, and its size was estimated to be approximately 50 kb (Figure 1).

2) Morphological classification of isolated phage

According to PAH1-C and C-PAH6 is Ackermann classification, in the form of cotton were pre-classified into five bidet (myoviridae) and (family) kawoodo Bilal less (Caudovirales) neck (order) (Fig. 2). The tail length and width were 113 ± 6/124 ± 6 nm (mean ± SD) (n = 10) and 14 ± 3/18 ± 1 nm (n = 10) respectively and the head diameter was 41 ± 3/63 ± 3 nm (n = 10).

Aeromonas Hydropila  About PAH1 -C PAH6 - Of C  Effectiveness test

1) Host cell lysate test

Bacterial lysing activity of PAH1-C and PAH6-C was tested on cultures of Aeromonas hydropila AH16 (Fig. 3). Time to remain on Pseudomonas dihydro pillar AH16 cultures are not infected with PAH1-C and PAH6-C (control), OD 600 value was increased continuously during the incubation. However, during the 24 hours after infection with PAH1-C and PAH6-C, the viability of Aeromonas hydropila AH16 was significantly inhibited at MOIs of 0.01, 1, and 100.

2) Aeromonas Hydropila  Infected Experimental words  Therapeutic effect of phage through

The group inoculated with Aeromonas hydropila AH16 died within 3 days, and in the group treated with PAH1-C and PAH6-C, there was a delay or a decrease in mortality. In addition, it was found that PAH6-C was superior to PAH1-C in inhibiting aeromonas hydropila infectious disease (Fig. 4).

BRC KCTC12128BP 20120203 BRC KCTC12129BP 20120203

Claims (8)

To remain on kawoodo having a specific ability to kill Pseudomonas dihydro pillar (Aeromonas hydrophila) Bilal less Mai ohbiri to (Caudovirales Myoviridae ) accession number KCTC 12128BP or accession number KCTC 12129BP. A composition for inhibiting or killing aeromonas hydrophila propagation comprising a caudoviral myoviride accession number KCTC 12128BP or a caudoviral myoviride accession number KCTC 12129BP as an active ingredient. The composition for inhibiting or killing aeromonas hydrophila according to claim 2, wherein the aeromonas hydrophila is an antibiotic resistant strain or a multidrug resistant strain. A pharmaceutical composition for preventing or treating aeromonas hydrophila infectious disease comprising the caudoviral myoviride accession number KCTC 12128BP or the caudoviral myoviride accession number KCTC 12129BP as an active ingredient. The pharmaceutical composition of claim 4, wherein the Aeromonas hydrophilic infectious disease is sepsis, Aeromonas disease or ulcer. Antibiotics comprising a caudoviral myoviride accession number KCTC 12128BP or a caudoviral myoviride accession number KCTC 12129BP as an active ingredient. A disinfectant comprising a caudovirales myoviride accession number KCTC 12128BP or a caudovirales myoviride accession number KCTC 12129BP as an active ingredient. A feed additive for fish comprising a caudoviral myoviride accession number KCTC 12128BP or a caudoviral myoviride accession number KCTC 12129BP as an active ingredient.

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